Lysophospholipase

ABSTRACT

The inventors have isolated lysophospholipases from  Aspergillus  ( A. niger  and  A. oryzae ) having molecular masses of about 68 kDa and amino acid sequences of 600-604 amino acid residues. The novel lysophospholipases have only a limited homology to known amino acid sequences. The inventors also isolated genes encoding the novel enzymes and cloned them into  E. coli  strains.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.10/309,437, filed Dec. 4, 2002, which is a divisional of U.S.application Ser. No. 09/687,538, filed Oct. 13, 2000, now allowed, whichis a continuation of U.S. application Ser. No. 09/678,513, filed on Oct.3, 2000, now abandoned, and claims priority of Danish application no. PA1999 01473, filed Oct. 14, 1999, and U.S. provisional application SerNo. 60/160,572 filed. Oct. 20, 1999, the contents of which are fullyincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to lysophospholipases (LPL), methods ofusing and producing them, as well as nucleic acid sequences encodingthem.

BACKGROUND OF THE INVENTION

Lysophospholipases (EC 3.1.1.5) are enzymes that can hydrolyze2-lysophospholids to release fatty acid. They are known to be useful,e.g., for improving the filterability of an aqueous solution containinga starch hydrolysate, particularly a wheat starch hydrolysate (EP219,269).

N. Masuda et al., Eur. J. Biochem., 202, 783-787 (1991) describe an LPLfrom Penicillium notatum as a glycoprotein having a molecular mass of 95kDa and a published amino acid sequence of 603 amino acid residues. WO98/31790 and EP 808,903 describe LPL from Aspergillus foetidus andAspergillus niger, each having a molecular mass of 36 kDa and an aminoacid sequence of 270 amino acids.

JP-A 10-155493 describes a phospholipase A1 from Aspergillus oryzae. Themature protein has 269 amino acids.

SUMMARY OF THE INVENTION

The inventors have isolated lysophospholipases from Aspergillus (A.niger and A. oryzae) having molecular masses of about 68 kDa and aminoacid sequences of 600-604 amino acid residues. The novellysophospholipases have only a limited homology to known amino acidsequences. The inventors also isolated genes encoding the novel enzymesand cloned them into E. coli strains.

Accordingly, the invention provides a lysophospholipase which may be apolypeptide having an amino acid sequence as the mature peptide shown inone of the following or which can be obtained therefrom by substitution,deletion, and/or insertion of one or more amino acids, particularly bydeletion of 25-35 amino acids at the C-terminal:

SEQ ID NO: 2 (hereinafter denoted A. niger LLPL-1),

SEQ ID NO: 4 (hereinafter denoted A. niger LLPL-2),

SEQ ID NO: 6 (hereinafter denoted A. oryzae LLPL-1), or

SEQ ID NO: 8 (hereinafter denoted A. oryzae LLPL-2).

Further, the lysophospholipase of the invention may be a polypeptideencoded by the lysophospholipase encoding part of the DNA sequencecloned into a plasmid present in Escherichia coli deposit number DSM13003, DSM 13004, DSM 13082 or DSM 13083.

The lysophospholipase may also be an analogue of the polypeptide definedabove which:

i) has at least 70% homology with said polypeptide,

ii) is immunologically reactive with an antibody raised against saidpolypeptide in purified form,

iii) is an allelic variant of said polypeptide,

Finally, the phospholipase of the invention may be a polypeptide whichis encoded by a nucleic acid sequence which hybridizes under highstringency conditions with one of the following sequences or itscomplementary strand or a subsequence thereof of at least 100nucleotides:

nucleotides 109-1920 of SEQ ID NO: 1 (encoding A. niger LLPL-1),

nucleotides 115-1914 of SEQ ID NO: 3 (encoding A. niger LLPL-2),

nucleotides 70-1881 of SEQ ID NO: 5 (encoding A. oryzae LLPL-1), or

nucleotides 193-2001 of SEQ ID NO: 7 (encoding A. oryzae LLPL-2).

The nucleic acid sequence of the invention may comprise a nucleic acidsequence which encodes any of the lysophospholipases described above, orit may encode a lysophospholipase and comprise:

a) the lysophospholipase encoding part of the DNA sequence cloned into aplasmid present in Escherichia coli DSM 13003, DSM 13004, DSM 13082 orDSM 13083 (encoding A. niger LLPL-1, A. niger LLPL-2, A. oryzae LLPL-1and A. oryzae LLPL-2, respectively),

b) the DNA sequence shown in SEQ ID NO: 1, 3, 5 or 7 (encoding A. nigerLLPL-1, A. niger LLPL-2, A. oryzae LLPL-1 and A. oryzae LLPL-2,respectively), or

c) an analogue of the DNA sequence defined in a) or b) which

i) has at least 70% homology with said DNA sequence, or

ii) hybridizes at high stringency with said DNA sequence, itscomplementary strand or a subsequence thereof.

Other aspects of the invention provide a recombinant expression vectorcomprising the DNA sequence, and a cell transformed with the DNAsequence or the recombinant expression vector.

A comparison with full-length prior-art sequences shows that the matureamino acid sequences of the invention have 60-69% homology with LPL fromPenicillium notatum (described above), and the corresponding DNAsequences of the invention show 63-68% homology with that of P. notatumLPL.

A comparison with published partial sequences shows that an expressedsequence tag (EST) from Aspergillus nidulans (GenBank M965865) of 155amino acid residues can be aligned with the mature A. oryzae LLPL-2 ofthe invention (604 amino acids) with a homology of 79%.

DETAILED DESCRIPTION OF THE INVENTION Genomic DNA Source

Lysophospholipases of the invention may be derived from strains ofAspergillus, particularly strains of A. niger and A. oryzae, usingprobes designed on the basis of the DNA sequences in this specification.

Strains of Escherichia coli containing genes encoding lysophospholipasewere deposited by the inventors under the terms of the Budapest Treatywith the DSMZ—Deutsche Sammiung von Microorganismen und ZellkulturenGmbH, Mascheroder Weg 1b, D-38124 Braunschweig DE as follows:Designation of Accession Source organism lysophospholipase number Datedeposited A. niger LLPL-1 DSM 13003 18 Aug. 1999 A. niger LLPL-2 DSM13004 18 Aug. 1999 A. oryzae LLPL-1 DSM 13082  8 Oct. 1999 A. oryzaeLLPL-2 DSM 13083  8 Oct. 1999

C-terminal Deletion

The lysophospholipase may be derived from the mature peptide shown inSEQ ID NOS: 2, 4, 6 or 8 by deletion at the C-terminal to remove the cosite residue while preserving the lysophospholipase activity. The ω siteresidue is described in Yoda et al. Biosci. Biotechnol. Biochem. 64,142-148, 2000, e.g. S577 of SEQ ID NO: 4. Thus, the C-terminal deletionmay particularly consist of 25-35 amino acid residues.

A lysophospholipase with a C-terminal deletion may particularly beproduced by expression in a strain of A. oryzae.

Properties of Lysophospholipase

The lysophospholipase of the invention is able to hydrolyze fatty acylgroups in lysophospholipid such as lyso-lecithin (Enzyme Nomenclature EC3.1.1.5). It may also be able to release fatty acids from intactphospholipid (e.g. lecithin).

Recombinant Expression Vector

The expression vector of the invention typically includes controlsequences encoding a promoter, operator, ribosome binding site,translation initiation signal, and, optionally, a selectable marker, atranscription terminator, a repressor gene or various activator genes.The vector may be an autonomously replicating vector, or it may beintegrated into the host cell genome.

Production by Cultivation of Transformant

The lysophospholipase of the invention may be produced by transforming asuitable host cell with a DNA sequence encoding the phospholipase,cultivating the transformed organism under conditions permitting theproduction of the enzyme, and recovering the enzyme from the culture.

The host organism is preferably a eukaryotic cell, in particular afungal cell, such as a yeast cell or a filamentous fungal cell, such asa strain of Aspergillus, Fusarium, Trichoderma or Saccharomyces,particularly A. niger, A. oryzae, F. graminearum, F. sambucinum, F.cerealis or S. cerevisiae, e.g. a glucoamylase-producing strain of A.niger such as those described in U.S. Pat. No. 3,677,902 or a mutantthereof. The production of the lysophospholipase in such host organismsmay be done by the general methods described in EP 238,023 (NovoNordisk), WO 96/00787 (Novo Nordisk) or EP 244,234 (Alko).

Hybridization

The hybridization is used to indicate that a given DNA sequence isanalogous to a nucleotide probe corresponding to a DNA sequence of theinvention. The hybridization conditions are described in detail below.

Suitable conditions for determining hybridization between a nucleotideprobe and a homologous DNA or RNA sequence involves presoaking of thefilter containing the DNA fragments or RNA to hybridize in 5×SSC(standard saline citrate) for 10 min, and prehybridization of the filterin a solution of 5×SSC (Sambrook et al. 1989), 5× Denhardt's solution(Sambrook et al. 1989), 0.5% SDS and 100 μg/ml of denatured sonicatedsalmon sperm DNA (Sambrook et al. 1989), followed by hybridization inthe same solution containing a random-primed (Feinberg, A. P. andVogelstein, B. (1983) Anal. Biochem. 132:6-13), ³²P-dCTP-labeled(specific activity >1×10⁹ cpm/μg) probe for 12 hours at approx. 45° C.The filter is then washed two times for 30 minutes in 2×SSC, 0.5% SDS ata temperature of at least 55° C., more preferably at least 60° C., morepreferably at least 65° C., even more preferably at least 70° C.,especially at least 75° C.

Molecules to which the oligonucleotide probe hybridizes under theseconditions are detected using a x-ray film.

Alignment and Homology

The lysophospholipase and the nucleotide sequence of the inventionpreferably have homologies to the disclosed sequences of at least 80%,particularly at least 90% or at least 95%, e.g. at least 98%.

For purposes of the present invention, alignments of sequences andcalculation of homology scores were done using a full Smith-Watermanalignment, useful for both protein and DNA alignments. The defaultscoring matrices BLOSUM50 and the identity matrix are used for proteinand DNA alignments respectively. The penalty for the first residue in agap is −12 for proteins and −16 for DNA, while the penalty foradditional residues in a gap is −2 for proteins and −4 for DNA.Alignment is from the FASTA package version v20u6 (W. R. Pearson and D.J. Lipman (1988), “Improved Tools for Biological Sequence Analysis”,PNAS 85:2444-2448, and W. R. Pearson (1990) “Rapid and SensitiveSequence Comparison with FASTP and FASTA”, Methods in Enzymology,183:63-98). Multiple alignments of protein sequences were done using“ClustalW” (Thompson, J. D., Higgins, D. G. and Gibson, T. J. (1994)CLUSTAL W: improving the sensitivity of progressive multiple sequencealignment through sequence weighting, positions-specific gap penaltiesand weight matrix choice. Nucleic Acids Research, 22:4673-4680).Multiple alignment of DNA sequences are done using the protein alignmentas a template, replacing the amino acids with the corresponding codonfrom the DNA sequence.

Lysophospholipase Activity (LLU)

Lysophospholipase activity is measured using egg yolk L-α-lysolecithinas the substrate with a NEFA C assay kit.

20 μl of sample is mixed with 100 μl of 20 mM sodium acetate buffer (pH4.5) and 100 μl of 1% L-α-lysolecithin solution, and incubated at 55° C.for 20 min. After 20 min, the reaction mixture is transferred to thetube containing 30 μl of Solution A in NEFA kit preheated at 37° C.After 10 min incubation at 37° C., 600 μl of Solution B in NEFA kit isadded to the reaction mixture and incubated at 37° C. for 10 min.Activity is measured at 555 nm on a spectrophotometer. One unit oflysophospholipase activity (1 LLU) is defined as the amount of enzymethat can increase the A550 of 0.01 per minute at 55° C.

Use of Lysophospholipase

The lysophospholipase of the invention can be used in any applicationwhere it is desired to hydrolyze the fatty acyl group(s) of aphospholipid or lyso-phospholipid, such as lecithin or lyso-lecithin.

As an example, the lysophospholipase of the invention can be used in thepreparation of dough, bread and cakes, e.g. to improve the elasticity ofthe bread or cake. Thus, the lysophospholipase can be used in a processfor making bread, comprising adding the lysophospholipase to theingredients of a dough, kneading the dough and baking the dough to makethe bread. This can be done in analogy with U.S. Pat. No. 4,567,046(Kyowa Hakko), JP-A 60-78529 (QP Corp.), JP-A 62-111629 (QP Corp.), JP-A63-258528 (QP Corp.) or EP 426211 (Unilever).

The lysophospholipase of the invention can also be used to improve thefilterability of an aqueous solution or slurry of carbohydrate origin bytreating it with the lysophospholipase. This is particularly applicableto a solution or slurry containing a starch hydrolysate, especially awheat starch hydrolysate since this tends to be difficult to filter andto give cloudy filtrates. The lysophospholipase may advantageously beused together with a beta-glucanase and/or a xylanase, e.g. as describedin EP 219,269 (CPC International).

The lysophospholipase of the invention can be used in a process forreducing the content of phospholipid in an edible oil, comprisingtreating the oil with the lysophospholipase so as to hydrolyze a majorpart of the phospholipid, and separating an aqueous phase containing thehydrolyzed phospholipid from the oil. This process is applicable to thepurification of any edible oil which contains phospholipid, e.g.vegetable oil such as soy bean oil, rape seed oil and sunflower oil. Theprocess can be conducted according to principles known in the art, e.g.in analogy with U.S. Pat. No. 5,264,367 (Metallgesellschaft, Röhm); K.Dahlke & H. Buchold, INFORM, 6 (12), 1284-91 (1995); H. Buchold, FatSci. Technol., 95 (8), 300-304 (1993); JP-A 2-153997 (Showa Sangyo); orEP 654,527 (Metallgesellschaft, Röhm).

EXAMPLES Materials and Methods

Methods

Unless otherwise stated, DNA manipulations and transformations wereperformed using standard methods of molecular biology as described inSambrook et al. (1989) Molecular cloning: A laboratory manual, ColdSpring Harbor lab., Cold Spring Harbor, N.Y.; Ausubel, F. M. et al.(eds.) “Current protocols in Molecular Biology”, John Wiley and Sons,1995; Harwood, C. R., and Cutting, S. M. (eds.) “Molecular BiologicalMethods for Bacillus”. John Wiley and Sons, 1990.

Enzymes

Enzymes for DNA manipulations (e.g. restriction endonucleases, ligasesetc.) are obtainable from New England Biolabs, Inc. and were usedaccording to the manufacturer's instructions.

Plasmids/Vectors

pT7Blue (Invitrogen, Netherlands)

pUC19 (Genbank Accession #: X02514)

pYES 2.0 (Invitrogen, USA).

Microbial Strains

E. coli JM109 (TOYOBO, Japan)

E. coli DH12α (GIBCO BRL, Life Technologies, USA)

Aspergillus oryzae strain IFO 4177 is available from Institute forFermentation, Osaka (IFO) Culture Collection of Microorganisms, 17-85,Juso-honmachi, 2-chome, Yodogawa-ku, Osaka 532-8686, Japan.

A. oryzae BECh-2 is described in Danish patent application PA 199901726. It is a mutant of JaL 228 (described in WO 98/12300) which is amutant of IFO 4177.

Reagents

NEFA test kit (Wako, Japan)

L-α-lysolecithin (Sigma, USA).

Media and Reagents

Cove: 342.3 g/L Sucrose, 20 ml/L COVE salt solution, 10 mM Acetamide, 30g/L noble agar.

Cove-2: 30 g/L Sucrose, 20 ml/L COVE salt solution, 10 mM, Acetamide, 30g/L noble agar.

Cove salt solution: per liter 26 g KCl, 26 g MgSO4-7aq, 76 g KH2PO4, 50ml Cove trace metals.

Cove trace metals: per liter 0.04 g NaB407-10aq, 0.4 g CuSO4-5aq, 1.2 gFeSO4-7aq, 0.7 g MnSO4-aq, 0.7 g Na2MoO2-2aq, 0.7 g ZnSO4-7aq.

AMG trace metals: per liter 14.3 g ZnSO4-7aq, 2.5 g CuSO4-5aq, 0.5 gNiCl2, 13.8 g FeSO4, 8.5 g MnSO4, 3.0 g citric acid.

YPG: 4 g/L Yeast extract, 1 g/L KH2PO4, 0.5 g/L MgSO4-7aq, 5 g/LGlucose, pH 6.0.

STC: 0.8 M Sorbitol, 25 mM Tris pH 8, 25 mM CaCl2.

STPC: 40% PEG4000 in STC buffer.

Cove top agarose: 342.3 g/L Sucrose, 20 ml/L COVE salt solution, 10 mMAcetamide, 10 g/L low melt agarose.

MS-9: per liter 30 g soybean powder, 20 g glycerol, pH 6.0.

MDU-pH5: per liter 45 g maltose-laq, 7 g yeast extract, 12 g KH2PO4, 1 gMgSO4-7aq, 2 g K2SO4, 0.5 ml AMG trace metal solution and 25 g2-morpholinoethanesulfonic acid, pH 5.0.

MLC: 40 g/L Glucose, 50 g/L Soybean powder, 4 g/L Citric acid, pH 5.0.

MU-1: 260 g/L Maltdextrin, 3 g/L MgSO4-7aq, 6 g/L K2SO4, 5 g/L KH2PO4,0.5 ml/L AMG trace metal solution, 2 g/L Urea, pH 4.5.

Example 1 Cloning and expression of LLPL-1 gene from A. niger

Transformation in Aspergillus strain

Aspergillus oryzae strain BECh-2 was inoculated to 100 ml of YPG mediumand incubated for16 hrs at 32° C. at 120 rpm. Pellets were collected andwashed with 0.6 M KCl, and resuspended 20 ml 0.6 M KCl containing acommercial β-glucanase product (Glucanex, product of Novo Nordisk A/S)at the concentration of 30 μ/ml. Cultures were incubated at 32° C. at 60rpm until protoplasts formed, then washed with STC buffer twice. Theprotoplasts were counted with a hematometer and resuspended in an8:2:0.1 solution of STC:STPC:DMSO to a final concentration of 2.5×10e7protoplasts/mI. About 3 μg of DNA was added to 100 μl of protoplastssolution, mixed gently and incubated on ice for 30 min. One ml of SPTCwas added and incubated 30 min at 37° C. After the addition of 10 ml of50° C. Cove top agarose, the reaction was poured onto Cove agar plate.Transformation plates were incubated at 32° C. for 5 days.

Preparation of a llP1 Probe

A strain of Aspergillus niger was used as a genomic DNA supplier.

PCR reactions on Aspergillus niger genome DNA was done with the primersHU175 (SEQ ID NO: 9) and HU176 (SEQ ID NO: 10) designed based upon thealignment several lysophospholipases from Penicillium and Neurospora sp.

Reaction components (1 ng /μl of genomic DNA, 250 mM dNTP each, primer250 nM each, 0.1 U/μl in Taq polymerase in 1× buffer (Roche Diagnostics,Japan)) were mixed and submitted for PCR under the following conditions.Step Temperature Time 1 94° C.  2 min 2 92° C.  1 min 3 55° C.  1 min 472° C.  1 min 5 72° C. 10 min 6  4° C. foreverSteps 2 to 4 were repeated 30 times.

The expected size, 1.0 kb fragment was gel-purified with QIA gelextraction kit (Qiagen, Germany) and ligated into a pT7Blue vector withligation high (TOYOBO, Japan). The ligation mixture was transformed intoE. coli JM109. The resultant plasmid (pHUda94) was sequenced andcompared to the Penicillium lysophospholipase, showing that a cloneencodes the internal part of the lysophospholipase.

Cloning of llpl-1 Gene

In order to clone the missing part of the lysophospholipase gene, agenomic restriction map as constructed by using the PCR fragment asprobes to a Southern blot of Aspergillus niger DNA digested with sevenrestriction enzymes, separately and probed with 1.0 kb fragment encodingpartial lysophospholipase from pHUda94.

A hybridized 4-6 kb SphI fragment was selected for a llpl-1 genesubclone.

For construction of a partial genomic library of Aspergillus niger, thegenomic DNA was digested with SphI and run on a 0.7% agarose gel. DNAwith a size between 4 to 6 kb was purified and cloned into pUC19pretreated SphI and BAP (Bacterial alkaline phosphatase). The sphIsub-library was made by transforming the ligated clones into E. coliDH12α cells. Colonies were grown on Hybond-N+ membranes (AmershamPharmacia Biotech, Japan) and hybridized to DIG-labelled (Non-radioisotope) 1.0 kb fragment from pHUda94.

Positive colonies were picked up and their inserts were checked by PCR.Plasmids elected colonies were prepared and sequenced revealing 5 kbSphI fragment were containing whole llpl-1 gene.

Expression of llpl-1 Gene in Aspergillus oryzae

The coding region of the LLPL-1 gene was amplified from genomic DNA ofan Asperniger niger strain by PCR with the primers HU188 (SEQ ID NO: 11)and HU189 (SEQ ID NO: 12) which included a EcoRV and a XhoI restrictionenzyme site, respectively.

Reaction components (1 ng/μl of genomic DNA, 250 mM dNTP each, primer250 nM each 0.1 U/μl in Taq polymerase in 1× buffer (Roche Diagnostics,Japan)) were mixed and submitted for PCR under the following conditions.Step Temperature time 1 94° C.  2 min 2 92° C.  1 min 3 55° C.  1 min 472° C.  2 min 5 72° C. 10 min 6  4° C. foreverSteps 2 to 4 were repeated 30 times.

The 2 kb fragment was gel-purified with QIA gel extraction kit andligated into a pT7Blue vector with Ligation high. The ligation mixturewas transformed into E. coli JM109. The resultant plasmid (pLLPL1) wassequenced. The pLLPL1 was confirmed that no changes had happen in theLLPL-1 sequences.

The pLLPL1 was digested with EcoRV and XhoI and ligated into the NruIand XhoI sites in an Aspergillus expression cassette (pCaHj483) whichhas Aspergillus niger neutral amylase promoter, Aspergillus nidulans TPIleader sequences, Aspergillus niger glucoamylase terminator andAspergillus nidulans amdS gene as a marker. The resultant plasmid wasnamed pHUda103.

The LLPL-1 expression plasmid, pHUda103, was digested with NotI andabout 6.1 kb DNA fragment containing Aspergillus niger neutral amylasepromoter, LLPL-1 coding region, Aspergillus niger glucoamylaseterminator and Aspergillus nidulans amdS gene was gel-purified with QIAgel extraction kit.

The 6.1 kb DNA fragment was transformed into Aspergillus oryzae BECh-2.The selected transformants were inoculated in 100 ml of MS-9 media andcultivated at 30° C. for 1 day. 3 ml of grown cell in MS-9 medium wasinoculated to 100 ml of MDU-pH5 medium and cultivated at 30° C. for 3days. The supernatant was obtained by centrifugation. The cell wasopened by mixed with the equal volume of reaction buffer (50 mM KPB-pH6.0) and glass-beads for 5 min on ice and debris was removed bycentrifugation.

The lysophospholipase productivity of selected transformants wasdetermined as the rate of hydrolysis of L-a-lysolecithin at pH 4.5 and55° C. measured in units per ml relative to the activity of the hoststrain, BECh-2 which is normalized to 1.0. The results shown in thetable below clearly demonstrate the absence of increasedlysophospholipase activity in supernatants and the presence of increasedlysophospholipase activity in cell free extracts. Yield (supernatant)Yield (Cell fraction) Strain Relative activity Relative activity BECh-21.0 1.0 LP3 1.0 4.5 1.0 4.0 LP8 1.0 6.5 1.0 5.5

Example 2 Cloning and Expression of LLPL-2 Gene from A. niger

Preparation of a llp2 Probe

The same strain of Aspergillus niger as in Example 1 was used as agenomic DNA supplier.

PCR reactions on Aspergillus niger genomic DNA was done with the primersHU212 (SEQ ID NO: 13) and HU213 (SEQ ID NO: 14) designed based uponamino acid sequences from purified lysophospholipase from AMG 400L(described in Example 4).

Reaction components (1 ng/μl of genomic DNA, 250 mM dNTP each, primer250 nM each, 0.1 U/μl in Taq polymerase in 1× buffer (Roche Diagnostics,Japan)) were mixed and submitted for PCR under the following conditions.Step Temperature Time 1 94° C.  2 min 2 92° C.  1 min 3 50° C.  1 min 472° C.  1 min 5 72° C. 10 min 6  4° C. foreverSteps 2 to 4 were repeated 30 times.

The expected size, 0.6 kb fragment was gel-purified with QIA gelextraction kit (Qiagen, Germany) and ligated into a pT7Blue vector withligation, high (TOYOBO, Japan). The ligation mixture was transformedinto E. coli JM109. The resultant plasmid (pHUda114) was sequenced andcompared to the Penicillium lysophospholipase, showing that a cloneencodes the internal part of the lysophospholipase.

Cloning of llpl-2 Gene

In order to clone the missing part of the lysophospholipase gene, agenomic restriction map was constructed by using the PCR fragment asprobes to a Southern blot of Aspergillus niger DNA digested with sevenrestriction enzymes, separately and probed with 1.0 kb fragment encodingpartial lysophospholipase from pHUda114.

A hybridized 4-6 kb XbaI fragment was selected for a llpl-2 genesubclone.

For construction of a partial genomic library of Aspergillus niger, thegenomic DNA was digested with XbaI and run on a 0.7% agarose gel. DNAwith a size between 4 to 6 kb was purified and cloned into pUC19pretreated XbaI and BAP (Bacterial alkaline phosphatase). The XbaIsub-library was made by transforming the ligated clones into E. coliDH12α cells. Colonies grown on Hybond-N+ membranes (Amersham PharmaciaBiotech, Japan) and hybridized to DIG-labelled (Non-radio isotope) 1.0kb fragment from pHUda114.

Positive colonies were picked up and their inserts were checked by PCR.Plasmids from selected colonies were prepared and sequenced revealing 5kb XbaI fragment were containing whole llpl-2 gene.

Expression of llpl-2 Gene in Aspergillus oryzae

The coding region of the LLPL-2 gene was amplified from genomic DNA ofan Aspergillus niger strain by PCR with the primers HU225 (SEQ ID NO:15) and HU226 (SEQ ID NO: 16) which included a BgIII and a PmeIrestriction enzyme site, respectively.

Reaction components (1 ng/μl of genomic DNA, 250 mM dNTP each, primer250 nM each, 0.1 U/μl in Taq polymerase in 1× buffer (Roche Diagnostics,Japan)) were mixed and submitted for PCR under the following conditions.Step Temperature time 1 94° C.  2 min 2 92° C.  1 min 3 55° C.  1 min 472° C.  2 min 5 72° C. 10 min 6  4° C. foreverStep 2 to 4 were repeated 30 times.

The 2 kb fragment was gel-purified with QIA gel extraction kit andligated into a pT7Blue vector with Ligation high. The ligation mixturewas transformed into E. coli JM109. The resultant plasmid (pLLPL2) wassequenced. The pLLPL2 was confirmed that no changes had happen in theLLPL-2 sequences.

The pLLPL2 was digested with BgIII and PmeI and ligated into the BamHIand NruI sites in the Aspergillus expression cassette pCaHj483 which hasAspergillus niger neutral amylase promoter, Aspergillus nidulans TPIleader sequences, Aspergillus niger glucoamylase terminator andAspergillus nidulans amdS gene as a marker. The resultant plasmid waspHUdal23.

The LLPL-2 expression plasmid, pHUdal23, was digested with NotI andabout 6.0 kb DNA fragment containing Aspergillus niger neutral amylasepromoter, LLPL-2 coding region, Aspergillus niger glucoamylaseterminator and Aspergillus nidulans amdS gene was gel-purified with QIAgel extraction kit.

The 6.0 kb DNA fragment was transformed into Aspergillus oryzae BECh-2.The selected transformants were inoculated in 100 ml of MS-9 media andcultivated at 30° C. for 1 day. 3 ml of grown cell in MS-9 medium wasinoculated to 100 ml of MDU-pH5 medium and cultivated cultivated at 30°C. for 4 days.

The supernatant was obtained by centrifugation. The cell was opened bymixed with the equal volume of reaction buffer (50 mM KPB-pH 6.0) andglass-beads for 5 min on ice and debris was removed by centrifugation.

The lysophospholipase productivity of selected transformants wasdetermined as in Example 1. The results shown in the table below clearlydemonstrate the absence of increased lysophospholipase activity insupernatants and the presence of increased lysophospholipase activity incell free extracts. Yield (supernatant) Yield (Cell fraction) StrainRelative activity Relative activity BECh-2 1.0 1.0 Fg-9 1.0 22.5 Eg-151.0 18.0 Fg-27 1.0 17.0 Fg-33 1.0 14.5

Example 3 Cloning and Expression of LLPL Genes from E. coli Clones

Each of the following large molecular weight lysophospholipase (LLPL)genes is cloned from the indicated E. coli clone as genomic DNAsupplier, and the gene is expressed in A. oryzae as described inExamples 1 and 2. E. coli clone LLPL DSM 13003 A. niger LLPL-1 DSM 13004A. niger LLPL-2 DSM 13082 A. oryzae LLPL-1 DSM 13083 A. oryzae LLPL-2

Example 4 Isolation of A. niger LLPL-2 from AMG 300L

Purification of LLPL-2 from AMG 300L

A commercially available glucoamylase preparation from A. niger (AMG300L, product of Novo Nordisk A/S) was diluted 10-fold with Milli-Qwater and subsequently added ammonium sulfate to 80% saturation. Thesolution was stirred 1 hour at 4° C. followed by centrifugation on anSorvall RC-3B centrifuge, equipped with a GSA rotor head (4500 rpm for35 min). The precipitate was discarded and the supernatant dialysedagainst 50 mM sodium acetate, pH 5.5. The dialysed solution was appliedto a Q-Sepharose (2.6×4 cm) column in 50 mM sodium acetate, pH 5.5 at aflow rate of 300 ml h⁻¹. The column was washed (10× column volume) andproteins were eluted using a linear gradient of 0-0.35 M NaCl in 50 mMsodium acetate, pH 5.5 at a flow rate of 300 ml h⁻¹. Fractionscontaining activity were pooled, concentrated on an Amicon cell (10 kDacutoff) to 2.5 ml and applied to Superdex 200 H/R (1.6×60 cm) in 0.2 mMsodium acetate, pH 5.5 by draining into the bed. Proteins were elutedisocratically at a flow rate of 30 ml h⁻¹. The purified enzyme showed aspecific activity of 86 LLU/mg.

SDS-PAGE analysis showed three protein bands at around 40, 80, and 120kDa. N-terminal sequencing of the first 23 amino acids revealed that theprotein bands at 40 and 120 kDa had identical sequences (shown at theN-terminal of SEQ ID NO: 4), whereas the protein band at 80 kDa wasshown to have the sequence shown as SEQ ID NO: 19. IEF analysis showedthat LLPL-2 had a pl of around 4.2.

Enzymatic Characterisation of LLPL-2

LLPL-2 was show to have a bell-shaped pH-activity profile with optimalactivity at pH 4.0. The temperature optimum was found at 50° C. Theenzyme activity was completely stable at pH 4.5 after up to 120 hoursincubation at pH 4.5 and 50° C. LLPL-2 is furthermore completely stableat 50° C., whereas a half-life of 84 hours was determined at 60° C.LLPL-2 was not found to be dependent upon addition of mineral salts likesodium or calcium.

Example 5 Identification and Sequencing of LLPL-1 and LLPL-2 Genes fromA. oryzae

Cultivation of A. oryzae

Aspergillus oryzae strain IFO 4177 was grown in two 20-liter labfermentors on a 10-liter scale at 34° C. using yeast extract anddextrose in the batch medium, and maltose syrup, urea, yeast extract,and trace metals in the feed. Fungal mycelia from the first labfermentor were harvested by filtering through a cellulose filter (poresize 7-11 microns) after 27 hours, 68.5 hours, 118 hours, and 139 hoursof growth. The growth conditions for the second fermentor were identicalto the first one, except for a slower growth rate during the first 20hours of fermentation. Fungal mycelia from the second lab fermentor wereharvested as above after 68.3 hours of growth. The harvested myceliawere immediately frozen in liquid N₂ and stored at −80° C.

The Aspergillus oryzae strain IFO 4177 was also grown in four 20-literlab fermentors on a 10-liter scale at 34° C. using sucrose in the batchmedium, and maltose syrup, ammonia, and yeast extract in the feed. Thefirst of the four fermentations was carried out at pH 4.0. The second ofthe four fermentations was carried out at pH 7.0 with a constant lowagitation rate (550 rpm) to achieve the rapid development of reductivemetabolism. The third of the four fermentations was carried out at pH7.0 under phosphate limited growth by lowering the amount of phosphateand yeast extract added to the batch medium. The fourth of the fourfermentations was carried out at pH 7.0 and 39° C. After 75 hours offermentation the temperature was lowered to 34° C. At 98 hours offermentation the addition of carbon feed was stopped and the culture wasallowed to starve for the last 30 hours of the fermentation. Fungalmycelial samples from the four lab fermentors above were then collectedas described above, immediately frozen in liquid N₂, and stored at −80°C.

Aspergillus oryzae strain IFO 4177 was also grown on Whatman filtersplaced on Cove-N agar plates for two days. The mycelia were collected,immediately frozen in liquid N₂, and stored at −80° C.

Aspergillus oryzae strain IFO 4177 was also grown at 30° C. in 150 mlshake flasks containing RS-2 medium (Kofod et aL, 1994, Journal ofBiological Chemistry 269: 29182-29189) or a defined minimal medium.Fungal mycelia were collected after 5 days of growth in the RS-2 mediumand 3 and 4 days of growth in the defined minimal medium, immediatelyfrozen in liquid N₂, and stored at −80° C.

Construction of Directional cDNA Libraries from Asperqillus oryzae

Total RNA was prepared by extraction with guanidinium thiocyanatefollowed by ultra-centrifugation through a 5.7 M CsCl cushion (Chirgwinet al., 1979, Biochemistry 18: 5294-5299) using the followingmodifications. The frozen mycelia were ground in liquid N₂ to a finepowder with a mortar and a pestle, followed by grinding in a precooledcoffee mill, and immediately suspended in 5 volumes of RNA extractionbuffer (4 M guanidinium thiocyanate, 0.5% sodium laurylsarcosine, 25 mMsodium citrate pH 7.0, 0.1 M β-mercaptoethanol). The mixture was stirredfor 30 minutes at room temperature and centrifuged (20 minutes at 10 000rpm, Beckman) to pellet the cell debris. The supernatant was collected,carefully layered onto a 5.7 M CsCI cushion (5.7 M CsCl, 10 mM EDTA, pH7.5, 0.1% DEPC; autoclaved prior to use) using 26.5 ml supernatant per12.0 ml of CsCl cushion, and centrifuged to obtain the total RNA(Beckman, SW 28 rotor, 25 000 rpm, room temperature, 24 hours). Aftercentrifugation the supernatant was carefully removed and the bottom ofthe tube containing the RNA pellet was cut off and rinsed with 70%ethanol. The total RNA pellet was transferred to an Eppendorf tube,suspended in 500 ml of TE, pH 7.6 (if difficult, heat occasionally for 5minutes at 65° C.), phenol extracted, and precipitated with ethanol for12 hours at -20° C (2.5 volumes of ethanol, 0.1 volume of 3M sodiumacetate pH 5.2). The RNA was collected by centrifugation, washed in 70%ethanol, and resuspended in a minimum volume of DEPC. The RNAconcentration was determined by measuring OD₂₆₀₂₈₀.

The poly(A)⁺ RNA was isolated by oligo(dT)-cellulose affinitychromatography (Aviv & Leder, 1972, Proceedings of the National Academyof Sciences USA 69: 1408-1412). A total of 0.2 g of oligo(dT) cellulose(Boehringer Mannheim, Indianapolis, Ind.) was preswollen in 10 ml of 1×of column loading buffer (20 mM Tris-Cl, pH 7.6, 0.5 M NaCl, 1 mM EDTA,0.1% SDS), loaded onto a DEPC-treated, plugged plastic column (Poly PrepChromatography Column, Bio-Rad, Hercules, Calif.), and equilibrated with20 ml of 1× loading buffer. The total RNA (1-2 mg) was heated at 65° C.for 8 minutes, quenched on ice for 5 minutes, and after addition of 1volume of 2× column loading buffer to the RNA sample loaded onto thecolumn. The eluate was collected and reloaded 2-3 times by heating thesample as above and quenching on ice prior to each loading. Theoligo(dT) column was washed with 10 volumes of 1× loading buffer, thenwith 3 volumes of medium salt buffer (20 mM Tris-Cl, pH 7.6, 0.1 M NaCl,1 mM EDTA, 0.1% SDS), followed by elution of the poly(A)⁺ RNA with 3volumes of elution buffer (10 mM Tris-Cl, pH 7.6, 1 mM EDTA, 0.05% SDS)preheated to 65° C., by collecting 500 μl fractions. The OD₂₆₀ was readfor each collected fraction, and the mRNA containing fractions werepooled and ethanol precipitated at −20° C. for 12 hours. The poly(A)⁺RNA was collected by centrifugation, resuspended in DEPC-DIW and storedin 5-10 mg aliquots at −80° C.

Double-stranded cDNA was synthesized from 5 μg of Aspergillus oryzae IFO4177 poly(A)⁺ RNA by the RNase H method (Gubler and Hoffman 1983, supra;Sambrook et al., 1989, supra) using a hair-pin modification. Thepoly(A)⁺RNA (5 μg in 5 μl of DEPC-treated water) was heated at 70° C.for 8 minutes in a pre-siliconized, RNase-free Eppendorf tube, quenchedon ice, and combined in a final volume of 50 il with reversetranscriptase buffer (50 mM Tris-Cl pH 8.3, 75 mM KCl, 3 mM MgCl₂, 10 mMDTT) containing 1 mM of dATP, dGTP and dTTP, and 0.5 mM of5-methyl-dCTP, 40 units of human placental ribonuclease inhibitor, 4.81μg of oligo(dT)₁₈-NotI primer and 1000 units of SuperScript II RNaseH—reverse transcriptase.

First-strand cDNA was synthesized by incubating the reaction mixture at45° C. for 1 hour. After synthesis, the mRNA:cDNA hybrid mixture was gelfiltrated through a Pharmacia MicroSpin S-400 HR spin column accordingto the manufacturer's instructions.

After the gel filtration, the hybrids were diluted in 250 μl of secondstrand buffer (20 mM Tris-Cl pH 7.4, 90 mM KCl, 4.6 mM MgCl₂, 10 mM(NH₄)₂SO₄, 0.16 mM βNAD⁺) containing 200 iM of each dNTP, 60 units of E.coli DNA polymerase I (Pharmacia, Uppsala, Sweden), 5.25 units of RNaseH, and 15 units of E. coli DNA ligase. Second strand cDNA synthesis wasperformed by incubating the reaction tube at 16° C. for 2 hours, and anadditional 15 minutes at 25° C. The reaction was stopped by addition ofEDTA to 20 mM final concentration followed by phenol and chloroformextractions.

The double-stranded cDNA was ethanol precipitated at −20° C. for 12hours by addition of 2 volumes of 96% ethanol and 0.2 volume of 10 Mammonium acetate, recovered by centrifugation, washed in 70% ethanol,dried (SpeedVac), and resuspended in 30 ml of Mung bean nuclease buffer(30 mM sodium acetate pH 4.6, 300 mM NaCl, 1 mM ZnSO₄, 0.35 mMdithiothreitol, 2% glycerol) containing 25 units of Mung bean nuclease.The single-stranded hair-pin DNA was clipped by incubating the reactionat 30° C. for 30 minutes, followed by addition of 70 ml of 10 mMTris-Cl, pH 7.5, 1 mM EDTA, phenol extraction, and ethanol precipitationwith 2 volumes of 96% ethanol and 0.1 volume 3 M sodium acetate pH 5.2on ice for 30 minutes.

The double-stranded cDNAs were recovered by centrifugation (20,000 rpm,30 minutes), and blunt-ended with T4 DNA polymerase in 30 μl of T4 DNApolymerase buffer (20 mM Tris-acetate, pH 7.9, 10 mM magnesium acetate,50 mM potassium acetate, 1 mM dithiothreitol) containing 0.5 mM of eachdNTP, and 5 units of T4 DNA polymerase by incubating the reactionmixture at +16° C. for 1 hour. The reaction was stopped by addition ofEDTA to 20 mM final concentration, followed by phenol and chloroformextractions and ethanol precipitation for 12 h at −20° C. by adding 2volumes of 96% ethanol and 0.1 volume of 3M sodium acetate pH 5.2.

After the fill-in reaction the cDNAs were recovered by centrifugation asabove, washed in 70% ethanol, and the DNA pellet was dried in aSpeedVac. The cDNA pellet was resuspended in 25 μl of ligation buffer(30 mM Tris-Cl, pH 7.8, 10 mM MgCl₂, 10 mM dithiothreitol, 0.5 mM ATP)containing 2 μg EcoRI adaptors (0.2 μg/μl, Pharmacia, Uppsala, Sweden)and 20 units of T4 ligase by incubating the reaction mix at 16° C. for12 hours. The reaction was stopped by heating at 65° C. for 20 minutes,and then placed on ice for 5 minutes. The adapted cDNA was digested withNotI by addition of 20 μl autoclaved water, 5 μl of 10×NotI restrictionenzyme buffer and 50 units of NotI, followed by incubation for 3 hoursat 37° C. The reaction was stopped by heating the sample at 65° C. for15 minutes. The cDNAs were size-fractionated by agarose gelelectrophoresis on a 0.8% SeaPlaque GTG low melting temperature agarosegel (FMC, Rockland, Me.) in 1×TBE (in autoclaved water) to separateunligated adaptors and small cDNAs. The gel was run for 12 hours at 15V, and the cDNA was size-selected with a cut-off at 0.7 kb by cuttingout the lower part of the agarose gel. Then a 1.5% agarose gel waspoured in front of the cDNA-containing gel, and the double-strandedcDNAs were concentrated by running the gel backwards until it appearedas a compressed band on the gel. The cDNA-containing gel piece was cutout from the gel and the cDNA was extracted from the gel using the GFXgel band purification kit (Amersham, Arlington Heights, Ill.) asfollows. The trimmed gel slice was weighed in a 2 ml Biopure Eppendorftube, then 10 ml of Capture Buffer was added for each 10 mg of gelslice, the gel slice was dissolved by incubation at 60° C. for 10minutes, until the agarose was completely solubilized, the sample at thebottom of the tube by brief centrifugation. The melted sample wastransferred to the GFX spin column placed in a collection tube,incubated at 25° C. for 1 minite, and then spun at full speed in amicrocentrifuge for 30 seconds. The flow-through was discarded, and thecolumn was washed with 500 μl of wash buffer, followed by centrifugationat full speed for 30 seconds. The collection tube was discarded, and thecolumn was placed in a 1.5 ml Eppendorf tube, followed by elution of thecDNA by addition of 50 μl of TE pH 7.5 to the center of the column,incubation at 25° C. for 1 minute, and finally by centrifugation for 1minute at maximum speed. The eluted cDNA was stored at −20° C. untillibrary construction.

A plasmid DNA preparation for a EcoRI-NotI insert-containing pYES2.0cDNA clone, was purified using a QIAGEN Tip-100 according to themanufacturer's instructions (QIAGEN, Valencia, Calif. A total of 10 mgof purified plasmid DNA was digested to completion with NotI and EcoRIin a total volume of 60 il by addition of 6 ml of 10×NEBuffer for EcoRI(New England Biolabs, Beverly, Mass.), 40 units of NotI, and 20 units ofEcoRI followed by incubation for 6 hours at 37° C. The reaction wasstopped by heating the sample at 65° C. for 20 minutes. The digestedplasmid DNA was extracted once with phenol-chloroform, then withchloroform, followed by ethanol precipitation for 12 hours at −20° C. byadding 2 volumes of 96% ethanol and 0.1 volume of 3 M sodium acetate pH5.2. The precipitated DNA was resuspended in 25 ml of 1×TE pH 7.5,loaded on a 0.8% SeaKem agarose gel in 1×TBE, and run on the gel for 3hours at 60 V. The digested vector was cut out from the gel, and the DNAwas extracted from the gel using the GFX gel band purification kit(Amersham-Pharmacia Biotech, Uppsala, Sweden) according to themanufacturer's instructions. After measuring the DNA concentration byOD₂₆₀₂₈₀, the eluted vector was stored at −20° C. until libraryconstruction.

To establish the optimal ligation conditions for the cDNA library, fourtest ligations were carried out in 10 il of ligation buffer (30 mMTris-Cl pH 7.8, 10 mM MgCl₂, 10 mM DTT, 0.5 mM ATP) containing 7 μl ofdouble-stranded cDNA, (corresponding to approximately {fraction (1/10)}of the total volume in the cDNA sample), 2 units of T4 ligase, and 25ng, 50 ng and 75 ng of EcoRI-NotI cleaved pYES2.0 vector, respectively(Invitrogen). The vector background control ligation reaction contained75 ng of EcoRI-NotI cleaved pYES.0 vector without cDNA. The ligationreactions were performed by incubation at 16° C. for 12 hours, heated at65° C. for 20 minutes, and then 10 μl of autoclaved water was added toeach tube. One il of the ligation mixtures was electroporated (200 W,2.5 kV, 25 mF) to 40 μl electrocompetent E. coli DH10B cells (LifeTechnologies, Gaithersburg, Md.). After addition of 1 ml SOC to eachtransformation mix, the cells were grown at 37° C. for 1 hour, 50 μl and5 μl from each electroporation were plated on LB plates supplementedwith ampicillin at 100 μg per ml and grown at 37° C. for 12 hours. Usingthe optimal conditions, 18 Aspergillus oryzae IFO 4177 cDNA librariescontaining 1-2.5×10⁷ independent colony forming units was established inE. coli, with a vector background of ca. 1%. The cDNA library was storedas (1) individual pools (25,000 c.f.u./pool) in 20% glycerol at −80° C.;(2) cell pellets of the same pools at −20° C.; (3) Qiagen purifiedplasmid DNA from individual pools at −20° C. (Qiagen Tip 100); and (4)directional, double-stranded cDNA at −20° C.

Aspergillus oryzae EST (expressed sequence tag) Template Preparation

From each cDNA library described, transformant colonies were pickeddirectly from the transformation plates into 96-well microtiter dishes(QIAGEN, GmbH, Hilden Germany) which contained 200 μl TB broth (LifeTechnologies, Frederick Md.) with 100 μg ampicillin per ml. The plateswere incubated 24 hours with agitation (300 rpm) on a rotary shaker. Toprevent spilling and cross-contamination, and to allow sufficientaeration, the plates were covered with a microporous tape sheet AirPore(QIAGEN GmbH, Hilden Germany). DNA was isolated from each well using theQlAprep 96 Turbo kit (QIAGEN GmbH, Hilden Germany).

EST Sequencing and Analysis of Nucleotide Sequence Data of theAspergillus orvzae EST Library

Single-pass DNA sequencing of the Aspergillus oryzae ESTs was done witha Perkin-Elmer Applied Biosystems Model 377 XL Automatic DNA Sequencer(Perkin-Elmer Applied Biosystems, Inc., Foster City, Calif.) usingdye-terminator chemistry (Giesecke et aL, 1992, Journal of VirologyMethods 38: 47-60) and a pYES specific primer (Invitrogen, Carlsbad,Calif.). Vector sequence and low quality 3′ sequence were removed withthe pregap program from the Staden package (MRC, Cambridge, England).The sequences were assembled with TIGR Assembler software (Sutton etal., 1995, supra). The assembled sequences were searched with fastx3(see Pearson and Lipman, 1988, Proceedings of the National Academy ofScience USA 85: 2444-2448; Pearson, 1990, Methods in Enzymology 183:63-98) against a customized data-base consisting of protein sequencesfrom SWISSPROT, SWISSPROTNEW, TREMBL, TREMBLNEW, REMTREMBL, PDB andGeneSeqP. The matrix used was BL50.

Nucleotide Sequence Analysis

The nucleotide sequence of the lysophospholipase cDNA clones pEST204,and pEST1648 were determined from both strands by the dideoxychain-termination method (Sanger, F., Nicklen, S., and Coulson, A. R.(1977) Proc. Natl. Acad. Sci. U.S.A. 74, 5463-5467) using 500 ng ofQiagen-purified template (Qiagen, USA), the Taq deoxy-terminal cyclesequencing kit (Perkin-Elmer, USA), fluorescent labeled terminators and5 pmol of either pYES 2.0 polylinker primers (Invitrogen, USA) orsynthetic oligonucleotide primers. Analysis of the sequence data wasperformed according to Devereux et al., 1984 (Devereux, J., Haeberli,P., and Smithies, O. (1984) Nucleic Acids Res. 12, 387-395).

Example 6 Expression of LLPL-2 in Aspergillus oryzae and Aspergillusniger

Transformation in Asperqillus strain

Aspergillus oryzae strain BECh-2 and an Aspergillus niger strain wereeach inoculated to 100 ml of YPG medium and incubated for16 hrs at 32°C. at 120 rpm. Pellets were collected and washed with 0.6 M KCl, andresuspended 20 ml 0.6 M KCl containing Glucanex at the concentration of30 μl/ml. Cultures were incubated at 32° C. at 60 rpm until protoplastsformed, then washed with STC buffer twice. The protoplasts were countedwith a hematometer and resuspended in an 8:2:0.1 solution ofSTC:STPC:DMSO to a final concentration of 2.5×10e7 protoplasts/ml. About3 μg of DNA was added to 100 μl of protoplasts solution, mixed gentlyand incubated on ice for 30 min. One ml of SPTC was added and incubated30 min at 37° C. After the addition of 10 ml of 50° C. Cove top agarose,the reaction was poured onto Cove agar plate. Transformation plates wereincubated at 32° C. for 5 days.

Expression of LLPL-2 Gene in Asperqillus niqer

The coding region of the LLPL-2 gene was amplified from genomic DNA ofan Aspergillus niger strain by PCR with the primers HU225 (SEQ ID NO:15) and HU226 (SEQ ID NO: 16) which included a BgIII and a PmeIrestriction enzyme site, respectively.

Reaction components (1 ng /μl of genomic DNA, 250 mM dNTP each, primer250 nM each, 0.1 U/μl in Taq polymerase in 1× buffer (Roche Diagnostics,Japan)) were mixed and submitted for PCR under the following conditions.Step Temperature time 1 94° C.  2 min 2 92° C.  1 min 3 55° C.  1 min 472° C.  2 min 5 72° C. 10 min 6  4° C. foreverStep 2 to 4 were repeated 30 times.

The 2 kb fragment was gel-purified with QIA gel extraction kit andligated into a pT7Blue vector with Ligation high. The ligation mixturewas transformed into E. coli JM109. The resultant plasmid (pLLPL2) wassequenced, and it was confirmed that no changes had happened in theLLPL-2 sequences.

The pLLPL2 was digested with BgIII and PmeI and ligated into the BamHIand NruI sites in the Aspergillus expression cassette pCaHj483 which hasAspergillus niger neutral amylase promoter, Aspergillus nidulans TPIleader sequences, Aspergillus niger glucoamylase terminator andAspergillus nidulans amdS gene as a marker. The resultant plasmid wasnamed pHUdal23.

The LLPL-2 expression plasmid, pHUda123, was transformed into anAspergillus niger strain. Selected transformants were inoculated in 100ml of MLC media and cultivated at 30° C. for 2 days. 5 ml of grown cellin MLC medium was inoculated to 100 ml of MU-1 medium and cultivated at30° C. for 7 days.

Supernatant was obtained by centrifugation, and the lysophospholipaseactivity was measured as described above. The table below shows thelysophospholipase activity from of the selected transformants, relativeto the activity of the host strain, MBin114 which was normalized to 1.0.Yield (supernatant) Strain Relative activity MBin114 1.0 123N-33 63123N-38 150 123N-46 157 123N-48 101

The above results clearly demonstrate the presence of increasedlysophospholipase y in supernatants.

Expression and Secretion of C-terminal Deleted LLPL-2 Gene inAspergillus oryzae

LLPL-2 with the C-terminal deleted (LLPL-2-CD) was made from genomic DNAof a strain of A. niger by PCR with the primers HU219 (SEQ ID NO: 17)and HU244 (SEQ ID NO: 18) which included an EagI and a PmeI restrictionenzyme site, respectively.

Reaction components (1 ng /ml of genomic DNA, 250 mM dNTP each, primer250 nM 0.1 U/ ml in Taq polymerase in 1× buffer (Roche Diagnostics,Japan)) were mixed and submitted for PCR under the following conditions.Step Temperature time 1 94° C.   2 min 2 92° C.   1 min 3 55° C.   1 min4 72° C. 1.5 min 5 72° C.  10 min 6  4° C. foreverStep 2 to 4 were repeated 30 times.

The 1.3 kb fragment was digested with EagI and PmeI and ligated into theEagI and PmeI sites in the pLLPL-2 having LLPL-2 gene with Ligationhigh.(TOYOBO). The ligation mixture was transformed into E. coli JM109.The resultant plasmid (pHUda126) was sequenced to confirm thatnucleotides 115-1824 of SEQ ID NO: 3 were intact and that nucleotides1825-1914 of SEQ ID NO: 3 had been deleted, corresponding to aC-terminal deletion of amino acids S571-L600 of LLPL-2 (SEQ ID NO: 4).

The 2.0 kb fragment encoding LLPL-2-CD was obtained by digestingpHUda126 with BgIII and SmaI. The 2.0 kb fragment was gel-purified withthe QIA gel extraction kit and ligated into the BamHI and NruI sites inthe Aspergillus expression cassette pCaHj483 with Ligation high. Theligation mixture was transformed into E. coli JM109.

The resultant plasmid (pHUda128) for LLPL-2-CD expression cassette wasconstructed and transformed into the A. oryzae strain, BECh-2. Selectedtransformants were inoculated in 100 ml of MS-9 media and cultivated at30° C. for 1 day. 3 ml of grown cell in MS-9 medium was inoculated to100 ml of MDU-pH5 medium and cultivated cultivated at 30° C. for 3 days.

Supernatant was obtained by centrifugation, and the lysophospholipaseactivity was measured as described above. The table below shows thelysophospholipase activity from of the selected transformants, relativeto the activity of the host strain, BECh-2 which was normalized to 1.0.Yield (supernatant) Strain Relative activity BECh-2 1.0 128-3 9 128-9 7128-12 33 128-15 11

The above results clearly demonstrate the presence of increasedlysophospholipase activity in supernatants.

Example 7 Use of A. niger LLPL-2 in Filtration

Filtration performance was determined at 60° C. and pH 4.5 usingpartially hydrolyzed wheat starch, as follows: The wheat starchhydrolyzate (25 ml in a 100 ml flask) was mixed with LLPL-2 from Example4 at a dosage of 0.4 L/t dry matter and incubated 6 hours at 60° C.under magnetic stirring. A control was made without enzyme addition.After 6 hours incubation the hydrolyzate was decanted into a glass andleft to settle for 10 min at room temperature. The tendency of thesample to flocculate was determined by visual inspection and ranged asexcellent, good, fair, bad, or none. The filtration flux wassubsequently determined by running the sample through a filter (Whatmanno. 4) and measuring the amount of filtrate after 2, 5, and 10 min. Theclarity of the filtered sample was measured spectrophotometrically at720 nm. The flux of filtrate (ml) was as follows: Time Control LLPL-2  2min. 4 8  5 min. 8 13 10 min. 12 16

These results indicate that LLPL-2 showed a clear effect on thefiltration flux compared to a control sample. Furthermore a clearfiltrate was obtained by treatment with LLPL-2.

1-12. cancel.
 13. An isolated lysophospholipase, comprising: a) apolypeptide encoded by a lysophospholipase encoding part of the DNAsequence cloned into a plasmid present in Escherichia coli depositnumber DSM 13004; b) a polypeptide having an amino acid sequence ofamino acids 1-458 in SEQ ID NO: 4; c) an analogue of the polypeptidedefined in (a) or (b) which has at least 95% sequence homology with saidpolypeptide; or d) a polypeptide which is encoded by a nucleic acidsequence which hybridizes with a complementary strand of the nucleicacid sequence shown as nucleotides 115-1914 of SEQ ID NO:3 underhybridization conditions comprising prehybridizing in a solution of5×SSC, 5× Denhardt's solution, 0.5% SDS and 100 μg/ml of denaturedsonicated salmon sperm DNA, followed by hybridization in the samesolution for 12 hours at approx. 45° C., followed by washing in 2×SSC,0.5% SDS for 30 minutes at a temperature of at least 65° C.
 14. Thelysophospholipase of claim 12 which is native to a strain ofAspergillus.
 15. The lysophospholipase of claim 12, which is native to astrain of A. niger.
 16. The lysophospholipase of claim 12, comprising adeletion of 25-35 amino acids at the C-terminal end.
 17. Thelysophospholipase of claim 12, comprising a polypeptide that is encodedby a nucleic acid sequence which hybridizes with a complementary strandof the nucleic acid sequence shown as nucleotides 115-1914 of SEQ IDNO:3 under hybridization conditions comprising prehybridizing in asolution of 5×SSC, 5× Denhardt's solution, 0.5% SDS and 100 μg/ml ofdenatured sonicated salmon sperm DNA, followed by hybridization in thesame solution for 12 hours at approx. 45° C., followed by washing in 2 xSSC, 0.5% SDS for 30 minutes at a temperature of at least 65° C.
 18. Thelysophospholipase of claim 12, comprising a polypeptide that is encodedby a nucleic acid sequence which hybridizes with a complementary strandof the nucleic acid sequence shown as nucleotides 115-1914 of SEQ IDNO:3 under hybridization conditions comprising prehybridizing in asolution of 5×SSC, 5× Denhardt's solution, 0.5% SDS and 100 μg/ml ofdenatured sonicated salmon sperm DNA, followed by hybridization in thesame solution for 12 hours at approx. 45° C., followed by washing in2×SSC, 0.5% SDS for 30 minutes at a temperature of at least 70° C. 19.The lysophospholipase of claim 12, comprising a polypeptide that isencoded by a nucleic acid sequence which hybridizes with a complementarystrand of the nucleic acid sequence shown as nucleotides 115-1914 of SEQID NO:3 under hybridization conditions comprising prehybridizing in asolution of 5×SSC, 5× Denhardt's solution, 0.5% SDS and 100 pg/ml ofdenatured sonicated salmon sperm DNA, followed by hybridization in thesame solution for 12 hours at approx. 45° C., followed by washing in2×SSC, 0.5% SDS for 30 minutes at a temperature of at least 75° C. 20.The lysophospholipase of claim 12, comprising a polypeptide which has atleast 95% homology to the polypeptide encoded by a lysophospholipaseencoding part of the DNA sequence cloned into a plasmid present inEscherichia coli deposit number DSM 13004 or to the polypeptide havingan amino acid sequence of amino acids 1-458 in SEQ ID NO:
 4. 21. Thelysophospholipase of claim 12, comprising a polypeptide which has atleast 98% homology to the polypeptide encoded by a lysophospholipaseencoding part of the DNA sequence cloned into a plasmid present inEscherichia coli deposit number DSM 13004 or to the polypeptide havingan amino acid sequence of amino acids 1-458 in SEQ ID NO:
 4. 22. Thelysophospholipase of claim 12, comprising a polypeptide encoded by alysophospholipase encoding part of the DNA sequence cloned into aplasmid present in Escherichia coli deposit number DSM
 13004. 23. Thelysophospholipase of claim 12, comprising a polypeptide having an aminoacid sequence comprising amino acids 1-458 in SEQ ID NO:
 4. 24. Aprocess for hydrolyzing fatty acyl groups in a phospholipid orlysophospholipid, comprising treating the phospholipid orlysophospholipid with the lysophospholipase of claim
 12. 25. A processfor improving the filterability of an aqueous solution or slurry ofcarbohydrate origin which contains phospholipid, which process comprisestreating the solution or slurry with the lysophospholipase of claim 12.26. The process of claim 28, wherein the solution or slurry contains astarch hydrolysate.
 27. The process of claim 28, wherein the solution orslurry contains a wheat starch hydrolysate.